Bottom Line:
Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient.Phosphoinositide-3 kinase selectively regulates the late barbed end transient.Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient.

ABSTRACTThe epidermal growth factor (EGF)-induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF.

fig9: PLC activity is required for directional protrusion in response to an EGF source. (A) The response of a control (U73343) cell (top) and of a PLC-inhibited (U73122) cell (bottom) to an EGF microneedle (the white triangle represents the position of the tip of the needle, and the white arrowhead indicates the direction of protrusion). (B) The quantitation of membrane protrusion at: (a) the front of cells (corresponding to the protrusion along the front axis formed between the cell centroid and the tip of the pipette), closed diamonds represent control and open diamonds PLC-inhibited cells; (b) the side of cells (corresponding to the axis that forms a 90° angle with the front axis), closed triangles represent control and open triangles PLC-inhibited cells; (c) the back of cells (corresponding to the axis that forms a 180° angle with the front axis), closed circles represent control and open circles PLC-inhibited cells. Error bars are SEM values of the averages of 15 cells, in each group, pooled from at least three independent experiments.

Mentions:
To determine the involvement of the PLC-dependent early polymerization transient in chemotaxis, in carcinoma cells, we used a micropipette chemotaxis assay. An EGF-filled pipette was placed in the proximity of quiescent cells, and membrane protrusion was monitored in time-lapse. Control cells, either untreated (not depicted) or treated with the inactive isoform of the PLC inhibitor (Fig. 9 A), showed an immediate and oriented protrusion toward the pipette (Fig. 9 B). The protrusion toward the pipette was defined as the “front protrusion,” which started almost directly after the introduction of the pipette and continued progressing after the pipette was removed (90 s later) until it reached a plateau in 5 min (Fig. 9 B). The kinetics and extent of the front protrusion is similar to those observed in the global EGF upshift experiments (Fig. 5). In contrast, the back and the side of the control cells (the sides not facing the pipette), did not show any protrusion (Fig. 9 B), and retraction was observed ∼10 min after stimulation (not depicted), corresponding to polarization and onward movement of the cell toward the original position of the pipette.

fig9: PLC activity is required for directional protrusion in response to an EGF source. (A) The response of a control (U73343) cell (top) and of a PLC-inhibited (U73122) cell (bottom) to an EGF microneedle (the white triangle represents the position of the tip of the needle, and the white arrowhead indicates the direction of protrusion). (B) The quantitation of membrane protrusion at: (a) the front of cells (corresponding to the protrusion along the front axis formed between the cell centroid and the tip of the pipette), closed diamonds represent control and open diamonds PLC-inhibited cells; (b) the side of cells (corresponding to the axis that forms a 90° angle with the front axis), closed triangles represent control and open triangles PLC-inhibited cells; (c) the back of cells (corresponding to the axis that forms a 180° angle with the front axis), closed circles represent control and open circles PLC-inhibited cells. Error bars are SEM values of the averages of 15 cells, in each group, pooled from at least three independent experiments.

Mentions:
To determine the involvement of the PLC-dependent early polymerization transient in chemotaxis, in carcinoma cells, we used a micropipette chemotaxis assay. An EGF-filled pipette was placed in the proximity of quiescent cells, and membrane protrusion was monitored in time-lapse. Control cells, either untreated (not depicted) or treated with the inactive isoform of the PLC inhibitor (Fig. 9 A), showed an immediate and oriented protrusion toward the pipette (Fig. 9 B). The protrusion toward the pipette was defined as the “front protrusion,” which started almost directly after the introduction of the pipette and continued progressing after the pipette was removed (90 s later) until it reached a plateau in 5 min (Fig. 9 B). The kinetics and extent of the front protrusion is similar to those observed in the global EGF upshift experiments (Fig. 5). In contrast, the back and the side of the control cells (the sides not facing the pipette), did not show any protrusion (Fig. 9 B), and retraction was observed ∼10 min after stimulation (not depicted), corresponding to polarization and onward movement of the cell toward the original position of the pipette.

Bottom Line:
Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient.Phosphoinositide-3 kinase selectively regulates the late barbed end transient.Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient.

ABSTRACTThe epidermal growth factor (EGF)-induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF.